Functional investigation of transmembrane helix 3 in H+- translocating pyrophosphatase

Ching Hung Lee, Yen Wei Chen, Yun Tzu Huang, Yih Jiuan Pan, Chien Hsien Lee, Shih Ming Lin, Lin Kun Huang, Yueh Yu Lo, Yu Fen Huang, Yu Di Hsu, Shih Chung Yen, Jenn Kang Hwang, Rong Long Pan

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

H+-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH+-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H +-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H+-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH+-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K+-mediated stimulation of H+-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase.

Original languageEnglish
Pages (from-to)959-966
Number of pages8
JournalJournal of Membrane Biology
Volume246
Issue number12
DOIs
Publication statusPublished - 2013 Nov 29

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Physiology
  • Cell Biology

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